Recombinant fsh composition for treatment of infertility

ABSTRACT

Preparations including FSH, for example recombinant FSH, for use in the treatment of infertility.

CROSS-REFERENCE TO RELATED APPLICANTS

The present application is a continuation of U.S. application Ser. No.15/566,657, filed Oct. 13, 2017 (now U.S. Pat. No. 10,660,938), which isthe U.S. National Stage of International Application PCT/EP2016/058358,filed Apr. 15, 2016, and claims priority to European Patent ApplicationNo. 15164043.0, filed Apr. 17, 2015.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety.

The present invention relates to compositions and pharmaceuticalproducts for the treatment of infertility.

Assisted reproductive technology (ART) techniques such as in vitrofertilisation (IVF) are well known. These ART techniques generallyrequire a step of controlled ovarian stimulation (COS), in which acohort of follicles is stimulated to full maturity. Standard COSregimens include administration of gonadotrophins, such as folliclestimulating hormone (FSH) alone or in combination with luteinisinghormone (LH) activity to stimulate follicular development, normally withadministration of a GnRH analogue prior to and/or during stimulation toprevent premature LH surge. The pharmaceutical compositions generallyused for COS include recombinant follicle stimulating hormone (rFSH),urinary derived FSH, recombinant FSH+LH preparations, urinary derivedmenotrophin [human menopausal gonadotrophin (hMG)] and highly purifiedhuman menopausal gonadotrophin (HP-hMG). IVF can be associated with arisk of ovarian hyperstimulation syndrome (OHSS), which can be lifethreatening in severe cases.

As indicated above, standard COS protocols generally involveadministration of FSH. The dose of FSH generally depends on a number offactors, including age, any previous response to FSH stimulation, basallevel of FSH, antral follicle count and more recently anti-Müllerianhormone (AMH). The clinician will expect ovarian multifolliculardevelopment in response to a given dose, together with a rise incirculating 17-β-estradiol.

If the response (ovarian multifollicular development, rise incirculating 17-β-estradiol) to a given dose is adequate or as expected,this indicates normal ovarian function, which is also referred to asnormal ovarian reserve. Patients who do not respond well to FSHstimulation produce few follicles, and consequently their 17-β-estradiollevels during stimulation rise slowly and reach comparatively lowlevels. These patients are referred to as “low responders”, and may besaid to have a diminished ovarian reserve. Several factors are believedto be involved in low response, including increasing age, pelvicadhesions, ovarian disease and immunological factors.

The ability to predict the response potential of women to controlledovarian stimulation (COS) may allow the development of individualisedCOS protocols. This could, for example, reduce the risk of OHSS in womenpredicted to have an excessive response to stimulation, improvepregnancy outcomes in women classed as poor responders, and/or lead toreduced dose of (and exposure to) FSH and therefore reduced cost oftherapy (and increased safety of therapy) in specific patients.

The serum concentration of anti-Müllerian hormone (AMH) is nowestablished as a reliable marker of ovarian reserve. Decreasing levelsof AMH are correlated with reduced ovarian response to gonadotrophinsduring COS. Further, high levels of AMH are a good predictor ofexcessive ovarian response, and an indicator of risk of OHSS.

In a preliminary study of women under 35 years old undergoing ART, theCONSORT dosing algorithm (incorporating basal FSH, BMI, age and AFC) wasused to predict the optimal FSH starting dose for COS in women at riskof developing OHSS (Olivennes et. al., 2009). Individualising the dosedid lead to adequate oocyte yield and good pregnancy rate. However,there were high rates of cancellations in the low dose group (75 IU FSH)due to inadequate response, and OHSS did occur in a significantproportion of the patients.

As indicated above, standard COS protocols may include administration ofFSH. FSH is naturally secreted by the anterior pituitary gland andfunctions to support follicular development and ovulation. FSH comprisesa 92 amino acid alpha sub-unit, also common to the other glycoproteinhormones LH and CG, and a 111 amino acid beta sub-unit unique to FSHthat confers the biological specificity of the hormone (Pierce andParsons, 1981). Each sub-unit is post translationally modified by theaddition of complex carbohydrate residues. Both subunits carry 2 sitesfor N-linked glycan attachment, the alpha sub-unit at amino acids 52 and78 and the beta sub-unit at amino acid residues 7 and 24 (Rathnam andSaxena, 1975, Saxena and Rathnam, 1976). FSH is thus glycosylated toabout 30% by mass (Dias and Van Roey. 2001. Fox et al. 2001).

FSH purified from post-menopausal human urine has been used for manyyears in infertility treatment; both to promote ovulation in naturalreproduction and to provide oocytes for assisted reproductiontechnologies. The currently approved recombinant FSH (rFSH) products forovarian stimulation, such as follitropin alfa (GONAL-F, Merck Serono/EMDSerono) and follitropin beta (PUREGON/FOLLISTIM, MSD/Schering-Plough),are derived from a Chinese Hamster Ovary (CHO) cell line. Currently, norFSH products from a human cell line are commercially available.

There is considerable heterogeneity associated with FSH preparationswhich relates to differences in the amounts of various isoforms present.Individual FSH isoforms exhibit identical amino acid sequences butdiffer in the extent to which they are post-translationally modified;particular isoforms are characterised by heterogeneity of thecarbohydrate branch structures and differing amounts of sialic acid (aterminal sugar) incorporation, both of which appear to influence thespecific isoform bioactivity.

Glycosylation of natural FSH is highly complex. The glycans in naturallyderived pituitary FSH can contain a wide range of structures that caninclude combinations of mono-, bi-, tri- and tetra-antennary glycans(Pierce and Parsons, 1981. Ryan et al., 1987. Baenziger and Green,1988). The glycans can carry further modifications: core fucosylation,bisecting glucosamine, chains extended with acetyl lactosamine, partialor complete sialylation, sialylation with α2,3 and α2,6 linkages, andsulphated galactosamine substituted for galactose (Dalpathado et al.,2006). Furthermore, there are differences between the distributions ofglycan structures at the individual glycosylation sites. A comparablelevel of glycan complexity has been found in FSH derived from the serumof individuals and from the urine of post-menopausal women (Wide et al.,2007).

The glycosylation of recombinant FSH products reflects the range ofglycosyl-transferases present in the host cell line. The commerciallyavailable rFSH products are derived from engineered Chinese hamsterovary cells (CHO cells). The range of glycan modifications in CHO cellderived rFSH are more limited than those found on the natural products.Examples of the reduced glycan heterogeneity found in CHO cell derivedrFSH include a lack of bisecting glucosamine and a reduced content ofcore fucosylation and acetyl lactosamine extensions (Hard et al., 1990).In addition, CHO cells are only able to add sialic acid using the α2,3linkage (Kagawa et al, 1988, Takeuchi et al, 1988, Svensson et al.,1990); CHO cell derived rFSH only includes α2,3-linked sialic acid anddoes not include α2,6-linked sialic acid.

Thus CHO cell derived FSH is different from naturally produced FSH (e.g.human Pituitary/ serum/ urinary FSH) which contains glycans with amixture of α2,3 and α2,6-linked sialic acid, with a predominance of theformer. Thus, recombinant proteins expressed using the CHO system willdiffer from their natural counterparts in their type of terminal sialicacid linkages. This is an important consideration in the production ofbiologicals for pharmaceutical use since the carbohydrate moieties maycontribute to the pharmacological attributes of the molecule.

The present applicants have developed a human derived recombinant FSHwhich is the subject of International Patent Application No.PCT/GB2009/000978, published as WO2009/127826A. Recombinant FSH with amixture of both α2,3 and α2,6-linked sialic acid was made by engineeringa human cell line to express both rFSH and α2,3 sialyltransferase. Theexpressed product is highly acidic and carries a mix of both α2,3- andα2,6-linked sialic acids; the latter provided by the endogenous sialyltransferase activity. It was found that the type of sialic acid linkage,α2,3- or α2,6-, can have a dramatic influence on biological clearance ofFSH. Recombinant FSH with a mixture of both α2,3 and α2,6-linked sialicacid has two advantages over rFSH expressed in conventional CHO cells:first the material is more highly sialylated due to the combinedactivities of the two sialyltransferases; and secondly the material moreclosely resembles the natural FSH. This is likely to be morebiologically appropriate compared to CHO cell derived recombinantproducts that have produce only α2,3 linked sialic acid (Kagawa et al,1988, Takeuchi et al, 1988, Svensson et al., 1990) and have decreasedsialic acid content (Ulloa-Aguirre et al. 1995., Andersen et al. 2004).

Recently, it has been suggested that the follicle-stimulating hormonereceptor or FSH receptor (FSHR) may be related to or involved indiminished ovarian reserve. The FSH receptor is a transmembrane receptorthat interacts with FSH. The FSH receptor is G-protein-coupled7-transmembrane receptor linked to adenylate cyclase, with a largeN-terminal ligand-binding domain and a C-terminal cytoplasmic tail richin serine and threonine residues as putitive phosphorylation sites. Itsactivation is necessary for the hormonal functioning of FSH. It has beenpostulated that mutations in the FSH receptor might lead to a diminishedovarian reserve. As well as mutations, FSH receptor variants (FSHreceptor polymorphisms) are found. Two such polymorphisms are located atposition 307 (Ala/Thr) and position 680 (Asn/Ser) in exon 10 of the FSHreceptor (FIG. 4). These are variant Ala or Thr in position 307, and Asnor Ser in position 680. These polymorphisms lead to three distinct FSHreceptor genotypes with regard to position 680: Asn/Asn, Asn/Ser andSer/Ser [see Simoni et al, Journal of Clinical Endocrinology andMetabolism, Vol 84, No. 2, 751-755 (1999), Falconer et al, Acta ObstetGynecol Scand 2005: 84: 806-811 (2005), and Loutradis et al, Journal ofAssisted Reproduction and Genetics, Vol. 23, No. 4, (April 2006)].

The present applicants have found that patients identified as having lowAMH [AMH level <15 pmol/L, who would generally be associated with a lowresponse], as well as having variant Ser/Ser at position 680 of the FSHreceptor, have a longer duration of FSH treatment, compared to patientswith low AMH and variant Asn/Asn or variant Asn/Ser at position 680 ofthe FSH receptor. An increased starting FSH dose to patients having lowAMH [AMH level <15 pmol/L, (e.g. 0.05 pmol/L to 14.9 pmol/L, e.g. 5.0pmol/L to 14.9 pmol/L)], as well as having variant Ser/Ser at position680 of the FSH receptor, may therefore be an alternative to avoid thelonger duration of FSH treatment. This allows tailoring of the dose ofFSH in specific patients identified as having specific AMH level, aswell as specific polymorphism at the FSHR. Administration of a higherstarting dose of FSH to patients having low AMH [AMH level <15 pmol/L,(e.g. 0.05 pmol/L to 14.9 pmol/L, e.g. 5.0 pmol/L to 14.9 pmol/L)], aswell as having variant Ser/Ser at position 680 of the FSH receptor, isadvantageous because it may provide increased probability of success (interms of pregnancy and/or live birth) and better predictability ofsuccess. Success is more likely if the patient has an adequate response(expected ovarian multifollicular development, rise in circulating17-β-estradiol) occurring within an ideal treatment window. Success isfurther enhanced if the response is within the centre of this treatmentwindow; that is, not too early in the window and not too late. Reductionof the duration of treatment in patients having low AMH and variantSer/Ser at position 680 of the FSH receptor (by increasing the doseabove 12 μg) may bring the response towards the centre of the treatmentwindow, with enhanced likelihood of success.

According to the present invention in a first aspect there is provided acomposition (e.g. a pharmaceutical composition) for use in the treatmentof infertility, the composition comprising 9 to 24 μg folliclestimulating hormone (FSH), wherein the composition is for (e.g. daily)administration to a patient identified as (e.g. selected as) havingvariant Ser/Ser at position 680 of the FSH receptor (prior totreatment). The composition may be for (e.g. daily) administration to apatient identified as (e.g. selected as) having variant Ser/Ser atposition 680 of the FSH receptor and identified as (e.g. selected as)having a serum AMH level <15 pmol/L (e.g. 0.05 pmol/L to 14.9 pmol/L)(prior to treatment). The composition may comprise 9 to 24 μg FSH, forexample 10 to 18 μg FSH, for example 12 to 16 μg FSH, for example 12 to15 μg FSH. The composition may comprise >12 μg FSH, for example 12.3 to24 μg FSH, for example 12.33 to 24 μg FSH, for example 12.67 to 24 μgFSH, for example 13 to 24 μg FSH, for example 13 to 16 μg FSH, forexample 13 to 15 μg FSH.

The composition (e.g. pharmaceutical composition) may comprise a dailydose of, or a daily dose equivalent to, the amounts of human derivedrFSH defined above, herein, and in the claims. The composition (e.g.pharmaceutical composition) may be for (daily) administration of FSHstarting on day one of treatment and continuing for six to sixteen days,for example seven to sixteen days, for example 8 to 16 days, for example8 to 13 days. The treatment of infertility may comprise a step ofidentifying (e.g. determining, e.g. measuring) the variant at position680 of the FSH receptor of the patient; and a step of administering thedose to a patient (identified as) having variant Ser/Ser at position 680of the FSH receptor. The treatment of infertility may comprise a step ofidentifying (e.g. determining, e.g. measuring) the serum AMH level ofthe patient, and administering the dose to a patient (identified as)having serum AMH level of <15 pmol/L (e.g. 0.05 pmol/L to 14.9 pmol/L).

The FSH may be recombinant FSH. The FSH may be recombinant FSH includingα2,3- and α2,6- sialylation. The FSH may be recombinant FSH includingα2,3- and α2,6- sialylation wherein 1 to 99% of the total sialylation isα2,6- sialylation and 99% to 1% of the total sialylation is α2,3-sialylation. The FSH may be recombinant FSH including α2,3- and α2,6-sialylation wherein 1 to 50% of the total sialylation is α2,6-sialylation and 50% to 99% of the total sialylation is α2,3-sialylation.The FSH may be recombinant FSH including α2,3- and α2,6-sialylationwherein 5 to 40% of the total sialylation is α2,6-sialylation and 60% to95% of the total sialylation is α2,3-sialylation. Preferably the FSH isa human cell line derived recombinant FSH.

According to the present invention in a further aspect there is provideda composition (e.g. a pharmaceutical composition) comprising folliclestimulating hormone (FSH) for use in the treatment of infertility in apatient having a serum AMH level <15 pmol/L (e.g. 0.05 pmol/L to 14.9pmol/L) and having variant Ser/Ser at position 680 of the FSH receptor,wherein the composition is to be administered at a dose of or equivalentto 9 to 24 μg recombinant FSH per day; and wherein the treatment ofinfertility comprises a step of identifying (e.g. determining) the serumAMH level of the patient; a step of identifying the variant at position680 of the FSH receptor of the patient; and a step of administering thecomposition to a patient (identified as) having a serum AMH level <15pmol/L (e.g. 0.05 pmol/L to 14.9 pmol/L) and variant Ser/Ser at position680 of the FSH receptor. The composition may comprise 9 to 24 μg FSH,for example 10 to 18 μg FSH, for example 12 to 16 μg FSH, for example 12to 15 μg FSH. The composition may comprise >12 μg FSH, for example 12.3to 24 μg FSH, for example 12.33 to 24 μg FSH, for example 12.67 to 24 μgFSH, for example 13 to 24 μg FSH, for example 13 to 16 μg FSH, forexample 13 to 15 μg FSH.

The composition (e.g. a pharmaceutical composition) may be for use inthe treatment of infertility in a patient having a serum AMH level <15pmol/L (e.g. 0.05 pmol/L to 14.9 pmol/L) and having variant Ser/Ser atposition 680 of the FSH receptor [wherein the treatment of infertilitycomprises a step of identifying (e.g. determining) the serum AMH levelof the patient; a step of identifying the variant at position 680 of theFSH receptor of the patient; and a step of administering the dose to apatient (identified as) having a serum AMH level <15 pmol/L (e.g. 0.05pmol/L to 14.9 pmol/L) and variant Ser/Ser at position 680 of the FSHreceptor].

The FSH may be recombinant FSH. The FSH may be recombinant FSH includingα2,3- and α2,6-sialylation. The FSH may be recombinant FSH includingα2,3- and α2,6-sialylation wherein 1 to 99% of the total sialylation isα2,6-sialylation and 99% to 1% of the total sialylation isα2,3-sialylation. The FSH may be recombinant FSH including α2,3- andα2,6-sialylation wherein 1 to 50% of the total sialylation isα2,6-sialylation and 50% to 99% of the total sialylation isα2,3-sialylation. The FSH may be recombinant FSH including α2,3- andα2,6-sialylation wherein 5 to 40% of the total sialylation isα2,6-sialylation and 60% to 95% of the total sialylation isα2,3-sialylation. Preferably the FSH is a human cell line derivedrecombinant FSH.

The dose provides an effective response while minimising risk of OHSS.

The doses above may be for treatment of infertility in the patient's(subject's) first stimulation protocol. It will be appreciated that forfurther stimulation cycles, the doses may be adjusted according toactual ovarian response in the first cycle.

The rFSH may be present as a single isoform or as a mixture of isoforms.

The applicants have devised “individualised” COS protocols whereinspecific doses of recombinant FSH are used to treat patients based ontheir specific AMH levels and FSHR single nucleotide polymorphism,thereby increasing the likelihood of adequate response to stimulation(e.g. in patients having a low response potential), and/or decreasedrisk of OHSS or other side effect.

The serum level of AMH may be determined (e.g. measured) by any methodknown in the art. As an example, the serum AMH level is measured usingthe AMH Gen-II enzyme linked immunosorbent assay, a kit (BeckmanCoulter, Inc., Webster, Tex.). This assay can detect AMH concentrationsgreater than 0.57 pmol/L with a minimum limit of quantitation of 1.1pmol/L. Other assays may be used. Herein, serum AMH values are generallyrecited in terms of pmol/L. This may be converted to ng/mL using theconversion equation 1 ng/ml AMH=7.1 pmol/L AMH. Thus, the compositionmay be for (e.g. daily) administration to a patient identified as (e.g.selected as) having a serum AMH level <15 pmol/L (e.g. 0.05 pmol/L to14.9 pmol/L) when measured using a Beckmann-Coulter Gen-II enzyme linkedimmunosorbent assay or a comparable AMH level measured by a differentmethod.

Herein the terms “patient” and “subject” are used interchangeably.

The composition (e.g. pharmaceutical composition) preferably comprises adaily dose of, or a daily dose equivalent to, the amounts of humanderived rFSH defined above, herein, and in the claims. The (daily) dosemay be an initial dose (i.e. it may be reduced, increased, or maintainedduring the treatment).

The patient identified as having variant Ser/Ser at position 680 of theFSH receptor may be identified by means well known in the art, forexample by identification of the allelic variant at position 680 of theFSH receptor following extraction of genomic DNA by methods well knownin the art [e.g. by means of a kit for extraction of genomic DNA fromblood, and subsequent DNA sequencing, as described in e.g. Gromoll etal, Methods, 21, 83-97 (2000), Simoni et al, Journal of ClinicalEndocrinology and Metabolism, Vol 84, No. 2, 751-755 (1999), Falconer etal, Acta Obstet Gynecol Scand 2005: 84: 806-811 (2005), and referencestherein, or DNA extraction followed by single strand conformationpolymorphism (SSCP followed by gel electrophoresis etc.), or by a PCRand RFLP method such as that set out in Loutradis et al, Journal ofAssisted Reproduction and Genetics, Vol. 23, No. 4, April 2006)]. Thus,the composition may be for (e.g. daily) administration to a patientidentified as (e.g. selected as) having variant Ser/Ser at position 680of the FSH receptor when measured by extraction of genomic DNA (e.g.from blood) and subsequent analysis by PCR and RFLP methods such as thatset out in Loutradis et al, Journal of Assisted Reproduction andGenetics, Vol. 23, No. 4, April 2006, or comparable method.

The composition (e.g. pharmaceutical composition) may be for (daily)administration of FSH starting on day one of treatment and continuingfor six to sixteen days, for example seven to sixteen days, for example8 to 16 days, for example 8 to 13 days. The composition (e.g.pharmaceutical composition) may be for administration 12 to 16, e.g. 13to 15, e.g. 14 days after administration of (e.g. after initiation ofadministration of, e.g. after initiation of daily administration of) aGnRH agonist (e.g. Synarel, Lupron, Decapeptyl). The composition (e.g.pharmaceutical composition) may be for administration with a GnRHagonist. The composition (e.g. pharmaceutical composition) may be foradministration prior to administration of a GnRH antagonist (e.g.ganirelix, cetrorelix), for example for administration five or six daysprior to administration of a GnRH antagonist. The composition (e.g.pharmaceutical composition) may be for administration with a GnRHantagonist. The composition (e.g. pharmaceutical composition) may be foradministration with a GnRH antagonist (e.g. ganirelix, cetrorelix)administered (e.g. daily) from day six of treatment. Preferably thecomposition (e.g. pharmaceutical composition) is for administrationprior to administration of a high (ovulatory) dose of hCG (for example4,000 to 11,000 IU hCG, e.g. 5,000 IU hCG, 10,000 IU hCG etc.; or 150 to350 microgram recombinant hCG, for example 250 microgram recombinanthCG) to induce final follicular maturation.

It will be appreciated that the composition may be for dosing atfrequencies more (or less) than daily, in which case the relevant doseswill be equivalent to the (daily) doses specified herein.

Herein the term “treatment of infertility” includes treatment ofinfertility by controlled ovarian stimulation (COS) or methods whichinclude a step or stage of controlled ovarian stimulation (COS), forexample Intra Uterine Insemination (IUI), in vitro fertilisation (IVF),or intracytoplasmic sperm injection (ICSI). The term “treatment ofinfertility” includes treatment of infertility by ovulation induction(OI) or by methods which include a step or stage of ovulation induction(OI). The term “treatment of infertility” includes treatment ofinfertility in a subject having tubal or unexplained infertility,including treatment of infertility in a subject having endometriosis,for example stage I or stage II endometriosis, and/or in a subjecthaving anovulatory infertility, for example WHO type II anovulatoryinfertility, and/or in a subject with a partner with male factorinfertility. The composition may be for (use in) the treatment ofinfertility (and/or for controlled ovarian stimulation) in a subjecthaving endometriosis, for example in a subject having stage I or stageII endometriosis, as defined by The American Society for ReproductiveMedicine (ASRM) classification system for the various stages ofendometriosis, (stage IV most severe; stage I least severe) [AmericanSociety for Reproductive Medicine. Revised American Society forReproductive Medicine classification of endometriosis: 1996. FertilSteril 1997; 67,817 821.].

The composition may be for (use in) the treatment of infertility (and/orfor controlled ovarian stimulation) in a subject having normal serum FSHlevel of 1 to 16 IU/L, for example 1 to 12 IU/L, in the early follicularphase.

The composition may be for (use in) the treatment of infertility (and/orfor controlled ovarian stimulation) in a subject identified as beingaged 18 to 42 years, for example 25 to 37 years. The product may be for(use in) the treatment of infertility (and/or for controlled ovarianstimulation) in a subject identified as having BMI >15 and BMI <38kg/m², for example a subject identified as having BMI >18 and BMI <25kg/m², for example a subject having BMI >20 and BMI <25 kg/m².

The rFSH may have a sialic acid content [expressed in terms of a ratioof moles of sialic acid to moles of protein] of 6 mol/mol or greater,for example between 6 mol/mol and 15 mol/mol, e.g between 8 mol/mol and14 mol/mol, e.g between 9 mol/mol and 14 mol/mol, for example between 10mol/mol and 14 mol/mol, e.g between 11 mol/mol and 14 mol/mol, e.gbetween 12 mol/mol and 14 mol/mol, e.g. between 12 mol/mol and 13mol/mol. The rFSH may be produced or expressed in a human cell line.

The FSH (rFSH) for use according to the invention may have 1% to 99% ofthe total sialylation being α2,3-sialylation. The rFSH may have 10% ormore of the total sialylation being α2,3-sialylation. For example, 20,30, 40, 50, 60, 70, 80 or 90% or more of the total sialylation may beα2,3-sialylation. The rFSH may preferably include α2,3-sialylation in anamount which is from 50 to 95% of the total sialylation, for examplefrom 50 to 70% of the total sialylation, for example from 60 to 69% ofthe total sialylation, for example from 63 to 67%, for example around65% of the total sialylation. The FSH (rFSH) for use according to theinvention may have 1% to 99% of the total sialylation beingα2,6-sialylation. The rFSH (or rFSH preparation) of the invention mayhave 5% or more, for example 5% to 99%, for example 5% to 50%, of thetotal sialylation being α2,6-sialylation. The rFSH may have 50% or lessof the total sialylation being α2,6-sialylation. The rFSH may preferablyinclude α2,6-sialylation in an amount which is from 5 to 50% of thetotal sialylation, for example from 10 to 50% of the total sialylation,for example from 31 to 38%, for example around 35% of the totalsialylation. By sialylation it is meant the amount of sialic residuespresent on the FSH carbohydrate structures. α2,3-sialylation meanssialylation at the 2,3 position (as is well known in the art) and α2,6sialylation at the 2,6 position (also well known in the art). Thus “% ofthe total sialylation may be α2,3 sialylation” refers to the % of thetotal number of sialic acid residues present in the FSH which aresialylated in the 2,3 position. The term “% of the total sialylationbeing α2,6-sialylation” refers to the % of the total number of sialicacid residues present in the FSH which are sialylated in the 2,6position.

The rFSH may have a sialic acid content (amount of sialylation per FSHmolecule) of (based on the mass of protein, rather than the mass ofprotein plus carbohydrate) of 6% or greater (e.g. between 6% and 15%,e.g. between 7% and 13%, e.g. between 8% and 12%, e.g. between 11% and15%, e.g. between 12% and 14%) by mass.

The rFSH may be produced or expressed in a human cell line, for examplea Per.C6 cell line, a HT1080 cell line etc.. This may simplify (andrender more efficient) the production method because manipulation andcontrol of e.g. the cell growth medium to retain sialylation may be lesscritical than with known processes. The method may also be moreefficient because there is little basic rFSH produced compared toproduction of known rFSH products; more acidic rFSH is produced andseparation/removal of basic FSH is less problematic. The rFSH may beproduced or expressed in a PER.C6® cell line, a PER.C6® derived cellline or a modified PER.C6® cell line. rFSH which is produced orexpressed in a human cell line (e.g. PER.C6® cell line, HT1080 cell lineetc.) will include some α2,6-linked sialic acids (α2,6 sialylation)provided by endogenous sialyl transferase activity [of the cell line]and will include some α2,3-linked sialic acids (α2,3 sialylation)provided by endogenous sialyl transferase activity. The cell line may bemodified using α2,3-sialyltransferase. The cell line may be modifiedusing α2,6-sialyltransferase. Alternatively or additionally, the rFSHmay include α2,6-linked sialic acids (α2,6 sialylation) provided byendogenous sialyl transferase activity [of the cell line]. Herein, theterm “human derived recombinant FSH” means recombinant FSH which isproduced or expressed in a human cell line (e.g. recombinant FSH made byengineering a human cell line).

The rFSH may be produced using α2,3- and/or α2,6-sialyltransferase. Inan example, rFSH is produced using α2,3-sialyltransferase. The rFSH mayinclude α2,6-linked sialic acids (α2,6 sialylation) provided byendogenous sialyl transferase activity.

The composition may be a pharmaceutical composition. The pharmaceuticalcomposition is for the treatment of infertility. The treatment ofinfertility may comprise assisted reproductive technologies (ART),ovulation induction or intrauterine insemination (IUI). Thepharmaceutical composition may be used, for example, in medicalindications where known FSH preparations are used.

The product or composition can be formulated into well-knowncompositions for any route of drug administration, e.g. oral, rectal,parenteral, transdermal (e.g. patch technology), intravenous,intramuscular, subcutaneous, intrasusternal, intravaginal,intraperitoneal, local (powders, ointments or drops) or as a buccal ornasal spray. A typical composition comprises a pharmaceuticallyacceptable carrier, such as aqueous solution, non toxic excipients,including salts and preservatives, buffers and the like, as described inRemington's Pharmaceutical Sciences fifteenth edition (Matt PublishingCompany, 1975), at pages 1405 to 1412 and 1461-87, and the nationalformulary XIV fourteenth edition (American Pharmaceutical Association,1975), among others.

Examples of suitable aqueous and non-aqueous pharmaceutical carriers,diluents, solvents or vehicles include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like),carboxymethylcellulose and suitable mixtures thereof, vegetable oils(such as olive oil), and injectible organic esters such as ethyl oleate.The compositions of the present invention also can contain additivessuch as but not limited to preservatives, wetting agents, emulsifyingagents, surfactants and dispersing agents. Antibacterial and antifungalagents can be included to prevent growth of microbes and includes, forexample, m-cresol, benzyl alcohol, paraben, chlorobutanol, phenol,sorbic acid, and the like. If a preservative is included, benzylalcohol, phenol and/or m-cresol are preferred; however, the preservativeis by no means limited to these examples. Furthermore, it may bedesirable to include isotonic agents such as sugars, sodium chloride,and the like. The product or composition may further comprise a saltcomprising a pharmaceutically acceptable alkali metal cation selectedfrom the group consisting of Na⁺- or K⁺- salts, or a combinationthereof. Preferably the salt is a Na+- salt, for example NaCl or Na₂SO₄.

Preferably the product or composition comprises recombinant FSH and oneor more of Polysorbate 20, L-methionine, phenol, disodium sulphate andsodium phosphate buffer.

In some cases, to effect prolonged action it is desirable to slow theabsorption of FSH (and other active ingredients, if present) fromsubcutaneous or intramuscular injection. This can be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of FSH then depends uponits rate of dissolution which, in turn, can depend upon crystal size andcrystalline form. Alternatively, delayed absorption of a parenterallyadministered FSH combination form is accomplished by dissolving orsuspending the FSH combination in an oil vehicle. Injectable depot formscan be made by forming microencapsule matrices of the FSH (and otheragents, if present) in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of FSH to polymerand the nature of the particular polymer employed, the rate of FSHrelease can be controlled. Examples of other biodegradable polymersinclude polyvinylpyrrolidone, poly(orthoesters), poly(anhydrides) etc.Depot injectable formulations are also prepared by entrapping the FSH inliposomes or microemulsions which are compatible with body tissues.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedor dispersed in sterile water or other sterile injectable medium justprior to use. Injectable formulations can be supplied in any suitablecontainer, e.g. vial, pre-filled syringe, injection cartridges, and thelike.

The product or composition may be formulated for single use or formultiple use (multiple dose). If the product or composition isformulated for multiple use, it is preferred that a preservative isincluded. If a preservative is included, benzyl alcohol, phenol and/orm-cresol are preferred; however, the preservative is by no means limitedto these examples. The single use or multiple use formulated product orcomposition may further comprise a salt comprising a pharmaceuticallyacceptable alkali metal cation selected from the group consisting ofNa⁺- or K⁺- salts, or a combination thereof. Preferably the salt is aNa+- salt, for example NaCl or Na₂SO₄.

The product or composition may be included in a container such as avial, prefilled cartridge (e.g. for single administration or multipleuse) or an injection device such as a “pen” for e.g. administration ofmultiple doses.

The product or composition may be a formulation (e.g. injectableformulation) including FSH (optionally with hCG, LH, LH activity etc.)The LH activity, if present, may originate from LH or human chorionicgonadotropin, hCG. If there is more than one active ingredient (i.e. FSHand e.g. hCG or LH) these may be suitable for administration separatelyor together. If administered separately, administration can besequential. The product can be supplied in any appropriate package. Forexample, a product can include a number of containers (e.g. pre-filledsyringes or vials) containing either FSH or hCG, or a combination (orcombination) of both FSH and hCG. The hCG may be recombinant hCG orurinary hCG. If the product includes a number of containers (e.g.pre-filled syringes or vials) containing FSH, e.g. recombinant FSH, eachcontainer may include the same amount of FSH. One or more containers mayinclude different amounts of FSH. The syringes or vials may be packagedin a blister package or other means to maintain sterility. Any productcan optionally contain instructions for using the FSH (and e.g. hCG ifpresent) formulations. The pH and exact concentration of the variouscomponents of the pharmaceutical composition are adjusted in accordancewith routine practice in this field. See GOODMAN and GILMAN's THEPHARMACOLOGICAL BASIS FOR THERAPEUTICES, 7^(th) ed. In a preferredembodiment, the compositions of the invention are supplied ascompositions for parenteral administration. General methods for thepreparation of the parenteral formulations are known in the art and aredescribed in REMINGTON; THE SCIENCE AND PRACTICE OF PHARMACY, supra, atpages 780-820. The parenteral compositions can be supplied in liquidformulation or as a solid which will be mixed with a sterile injectablemedium just prior to administration. In an especially preferredembodiment, the parenteral compositions are supplied in dosage unit formfor ease of administration and uniformity of dosage.

According to the present invention in a further aspect there is provideda method of treatment of infertility [e.g. infertility in a patienthaving a serum AMH level <15 pmol/L (e.g. 0.05 pmol/L to 14.9 pmol/L)and having variant Ser/Ser at position 680 of the FSH receptor] whereinthe treatment of infertility comprises (a) identifying (e.g.determining) the serum AMH level of the patient.

(b) identifying the variant at position 680 of the FSH receptor of thepatient; and (c) administering a dose of or equivalent to 9 to 24 μgrecombinant follicle stimulating hormone (FSH) per day to a patient(identified as) having a serum AMH level <15 pmol/L (e.g. 0.05 pmol/L to14.9 pmol/L) and variant Ser/Ser at position 680 of the FSH receptor.The composition may comprise 9 to 24 μg FSH, for example 10 to 18 μgFSH, for example 12 to 16 μg FSH, for example 12 to 15 μg FSH. Thecomposition may comprise >12 μg FSH, for example 12.3 to 24 μg FSH, forexample 12.33 to 24 μg FSH, for example 12.67 to 24 μg FSH, for example13 to 24 μg FSH, for example 13 to 16 μg FSH, for example 13 to 15 μgFSH.

The administration of FSH may be starting on day one of treatment andcontinuing for six to sixteen days, for example seven to sixteen days,for example 8 to 16 days, for example 8 to 13 days.

The FSH may be recombinant FSH. The FSH may be recombinant FSH includingα2,3- and α2,6-sialylation. The FSH may be recombinant FSH includingα2,3- and α2,6-sialylation wherein 1 to 99% of the total sialylation isα2,6-sialylation and 99% to 1% of the total sialylation isα2,3-sialylation. The FSH may be recombinant FSH including α2,3- andα2,6-sialylation wherein 1 to 50% of the total sialylation isα2,6-sialylation and 50% to 99% of the total sialylation isα2,3-sialylation. The FSH may be recombinant FSH including α2,3- andα2,6-sialylation wherein 5 to 40% of the total sialylation isα2,6-sialylation and 60% to 95% of the total sialylation isα2,3-sialylation. Preferably the FSH is a human cell line derivedrecombinant FSH.

The administration preferably comprises a daily dose of, or a daily doseequivalent to, the amount of FSH defined above and in the claims. The(daily) dose may be an initial dose (it may be reduced, increased, ormaintained during the treatment).

The method may be a method of treatment of infertility in the patient's(subject's) first stimulation protocol. It will be appreciated that forfurther stimulation cycles, the doses may be adjusted according toactual ovarian response in the first cycle.

According to the present invention in a further aspect there is provideda composition (e.g. a pharmaceutical composition) comprising FSH for usein the manufacture of a medicament for the treatment of infertility, thecomposition comprising 9 to 24 μg follicle stimulating hormone (FSH),wherein the medicament is for (e.g. daily) administration to a patientidentified as (e.g. selected as) having variant Ser/Ser at position 680of the FSH receptor (prior to treatment). The medicament may be for(e.g. daily) administration to a patient identified as (e.g. selectedas) having variant Ser/Ser at position 680 of the FSH receptor andidentified as (e.g. selected as) having a serum AMH level <15 pmol/L(e.g. 0.05 pmol/L to 14.9 pmol/L) (prior to treatment). The compositionmay comprise 9 to 24 μg FSH, for example 10 to 18 μg FSH, for example 12to 16 μg FSH, for example 12 to 15 μg FSH. The composition maycomprise >12 μg FSH, for example 12.3 to 24 μg FSH, for example 12.33 to24 μg FSH, for example 12.67 to 24 μg FSH, for example 13 to 24 μg FSH,for example 13 to 16 μg FSH, for example 13 to 15 μg FSH.

The present applicants also found that administration of FSH to patientsidentified as having low AMH [AMH level <15 pmol/L (e.g. 0.05 pmol/L to14.9 pmol/L), who would generally be associated with a low response], aswell as having variant Asn/Asn or variant Asn/Ser at position 680 of theFSH receptor, provides a good response in terms of folliculardevelopment. This is achieved with a reduced dosage of FSH and/orreduced duration of treatment, compared with treatment of patients withlow AMH and variant Ser/Ser at position 680 of the FSH receptor. Thisallows tailoring of the dose of FSH in specific patients identified ashaving specific AMH level, as well as these specific polymorphisms atthe FSHR. As set out below, the expected duration of stimulation forapatient having AMH level <15 pmol/L and variant Asn/Asn is 1.5 days lessthan the duration of stimulation required for a patient having AMH level<15 pmol/L and variant Ser/Ser (see FIG. 7). Accordingly, tailoring thedose to patients identified, prior to treatment, as having both AMHlevel <15 pmol/L and variant Asn/Asn (or both AMH level <15 pmol/L andvariant Asn/Ser) may allow considerable saving in terms ofpharmaceutical cost, as well as reduction in risk of potential sideeffects due to administration of a higher total dose of FSH than isrequired in these patients.

According to the present invention in a further aspect there is provideda composition (e.g. a pharmaceutical composition) for use in thetreatment of infertility, the composition comprising 10 to 12 μgfollicle stimulating hormone (FSH), wherein the composition is for (e.g.daily) administration to a patient identified as (e.g. selected as)having variant Asn/Asn or variant Asn/Ser at position 680 of the FSHreceptor (prior to treatment). The composition may be for (e.g. daily)administration to a patient identified as (e.g. selected as) havingvariant Asn/Asn or variant Asn/Ser at position 680 of the FSH receptorand identified as (e.g. selected as) having a serum AMH level <15 pmol/L(e.g. 0.05 pmol/L to 14.9 pmol/L) (prior to treatment). The compositionmay be for administration to a patient identified as (e.g. selected as)having variant Asn/Asn at position 680 of the FSH receptor andidentified as (e.g. selected as) having a serum AMH level <15 pmol/L(e.g. 0.05 pmol/L to 14.9 pmol/L) (prior to treatment). The compositionmay comprise 10 to <12 μg FSH, for example 10 to 11.9 μg FSH, forexample 11 to 11.9 μg FSH, for example 11.33 or 11.67 μg FSH.

The composition (e.g. pharmaceutical composition) may comprise a dailydose of, or a daily dose equivalent to, the amounts of human derivedrFSH defined above, herein, and in the claims. The composition (e.g.pharmaceutical composition) may be for (daily) administration of FSHstarting on day one of treatment and continuing for six to sixteen days,for example seven to sixteen days, for example 8 to 16 days, for example8 to 13 days. The treatment of infertility may comprise a step ofidentifying (e.g. determining, e.g. measuring) the variant at position680 of the FSH receptor of the patient; and a step of administering thedose to a patient (identified as) having variant Asn/Asn or variantAsn/Ser at position 680 of the FSH receptor. The treatment ofinfertility may comprise a step of identifying (e.g. determining, e.g.measuring) the serum AMH level of the patient, and administering thedose to a patient (identified as) having serum AMH level of <15 pmol/L(e.g. 0.05 pmol/L to 14.9 pmol/L).

According to the present invention in a further aspect there is provideda composition (e.g. a pharmaceutical composition) comprising folliclestimulating hormone (FSH) for use in the treatment of infertility in apatient having a serum AMH level <15 pmol/L (e.g. 0.05 pmol/L to 14.9pmol/L) and having variant Asn/Asn or variant Asn/Ser at position 680 ofthe FSH receptor, wherein the composition is to be administered at adose of or equivalent to 10 to 12 μg recombinant FSH per day; andwherein the treatment of infertility comprises a step of identifying(e.g. determining) the serum AMH level of the patient; a step ofidentifying the variant at position 680 of the FSH receptor of thepatient; and a step of administering the composition to a patient(identified as) having a serum AMH level <15 pmol/L (e.g. 0.05 pmol/L to14.9 pmol/L) and variant Asn/Asn or variant Asn/Ser at position 680 ofthe FSH receptor. The composition may comprise 10 to <12 μg FSH, forexample 10 to 11.9 μg FSH, for example 11 to 11.9 μg FSH, for example11.33 or 11.67 μg FSH.

The composition (e.g. a pharmaceutical composition) may be for use inthe treatment of infertility in a patient having a serum AMH level <15pmol/L (e.g. 0.05 pmol/L to 14.9 pmol/L) and having variant Asn/Asn atposition 680 of the FSH receptor [wherein the treatment of infertilitycomprises a step of identifying (e.g. determining) the serum AMH levelof the patient; a step of identifying the variant at position 680 of theFSH receptor of the patient; and a step of administering the dose to apatient (identified as) having a serum AMH level <15 pmol/L (e.g. 0.05pmol/L to 14.9 pmol/L) and variant Asn/Asn at position 680 of the FSHreceptor].

According to the present invention in a further aspect there is provideda method of treatment of infertility [e.g. infertility in a patienthaving a serum AMH level <15 pmol/L (e.g. 0.05 pmol/L to 14.9 pmol/L)and having variant Asn/Asn or variant Asn/Ser at position 680 of the FSHreceptor] wherein the treatment of infertility comprises (a) identifying(e.g. determining) the serum AMH level of the patient; (b) identifyingthe variant at position 680 of the FSH receptor of the patient; and (c)administering a dose of or equivalent to 10 to 12 μg recombinantfollicle stimulating hormone (FSH) per day to a patient (identified as)having a serum AMH level <15 pmol/L (e.g. 0.05 pmol/L to 14.9 pmol/L)and variant Asn/Asn or variant Asn/Ser at position 680 of the FSHreceptor. The composition may comprise 10 to <12 μg FSH, for example 10to 11.9 μg FSH, for example 11 to 11.9 μg FSH, for example 11.33 or11.67 μg FSH.

The administration of FSH may be starting on day one of treatment andcontinuing for six to thirteen days, for example seven to thirteen days,for example 8 to 13 days, for example 8 to 11 days.

According to the present invention in a further aspect there is provideda composition (e.g. a pharmaceutical composition) comprising FSH for usein the manufacture of a medicament for the treatment of infertility, thecomposition comprising 10 to 12 μg follicle stimulating hormone (FSH),wherein the medicament is for (e.g. daily) administration to a patientidentified as (e.g. selected as) having variant Asn/Asn or variantAsn/Ser at position 680 of the FSH receptor (prior to treatment). Themedicament may be for (e.g. daily) administration to a patientidentified as (e.g. selected as) having variant Asn/Asn or variantAsn/Ser at position 680 of the FSH receptor and identified as (e.g.selected as) having a serum AMH level <15 pmol/L (e.g. 0.05 pmol/L to14.9 pmol/L) (prior to treatment). The medicament may be foradministration to a patient identified as (e.g. selected as) havingvariant Asn/Asn at position 680 of the FSH receptor and identified as(e.g. selected as) having a serum AMH level <15 pmol/L (e.g. 0.05 pmol/Lto 14.9 pmol/L) (prior to treatment). The composition may comprise 10 to<12 μg FSH, for example 10 to 11.9 μg FSH, for example 11 to 11.9 μgFSH, for example 11.33 or 11.67 μg FSH.

It will be appreciated that the FSH, identification of patient's serumAMH level and variant at position 680 of the FSH receptor, etc. forthese aspects of the invention may be as for the other aspects of theinvention recited herein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described in more detail withreference to the attached drawings in which:

FIG. 1 shows a plasmid map of the pFSHalpha/beta expression vector;

FIG. 2 shows the α2,3-sialyltransferase (ST3GAL4) expression vector;

FIG. 3 shows the α2,6-sialyltransferase (ST6GAL1) expression vector;

FIG. 4 is a schematic diagram of the FSH Receptor, indicating theposition of polymorphisms at amino acid positions 307 and 680 of exon10, and position 29 in the promoter;

FIG. 5 shows the distribution of SNP Haplotypes at the FSH receptor genefor the 222 patients treated with FSH in the study of Example 8;

FIG. 6 is a table of results showing, for the full analysis set, theobserved duration of gonadotropin (FSH) treatment (days) and totalgonadotropin (FSH) dose delivered (μg) to patients/subjects having AMH<15 pmol/L in each of the three distinct FSH receptor genotypes withregard to position 680: Asn/Asn, Asn/Ser and Ser/Ser; and

FIG. 7 is a table of results showing, for the full analysis set, theexpected duration of gonadotropin (FSH) treatment (days) delivered topatients/subjects having AMH <15 pmol/L in each of the three distinctFSH receptor genotypes with regard to position 680: Asn/Asn, Asn/Ser andSer/Ser, adjusted for dose.

Sequence Selection Human FSH

The coding region of the gene for the FSH alpha polypeptide was used toaccording to Fiddes and Goodman. (1981). The sequence is banked asAH007338 and at the time of construction there were no other variants ofthis protein sequence. The sequence is referred herein as SEQ ID NO:1.

The coding region of the gene for FSH beta polypeptide was usedaccording to Keene et al (1989). The sequence is banked as NM_000510 andat the time of construction there were no other variants of this proteinsequence. The sequence is referred herein as SEQ ID NO: 2

Sialyltransferase

α2,3-Sialyltransferase—The coding region of the gene forbeta-galactoside alpha-2,3-sialyltransferase 4 (α2,3-sialyltransferase,ST3GAL4) was used according to Kitagawa and Paulson (1994). The sequenceis banked as L23767 and referred herein as SEQ ID NO: 3.

α2,6-Sialyltransferase—The coding region of the gene forbeta-galactosamide alpha-2,6-sialyltransferase 1(α2,6-sialyltransferase, ST6GAL1) was used according to Grundmann et al.(1990). The sequence is banked as NM_003032 and referred herein as SEQID NO: 4.

EXAMPLES Example 1 Construction of the FSH Expression Vector

The coding sequence of FSH alpha polypeptide (AH007338, SEQ ID NO: 1)and FSH beta polypeptide (NM_003032, SEQ ID NO: 2) were amplified by PCRusing the primer combinations FSHa-fw and FSHa-rev and FSHb-fw andFSHb-rec respectively.

FSHa-fw (SEQ ID NO: 9) 5′-CCAGGATCCGCCACCATGGATTACTACAGAAAAATATGC-3′FSHa-rev (SEQ ID NO: 10) 5′-GGATGGCTAGCTTAAGATTTGTGATAATAAC-3′ FSHb-fw(SEQ ID NO: 11) 5′-CCAGGCGCGCCACCATGAAGACACTCCAGTTTTTC-3′ FSHb-rev(SEQ ID NO: 12) 5′-CCGGGTTAACTTATTATTCTTTCATTTCACCAAAGG-3′

The resulting amplified FSH beta DNA was digested with the restrictionenzymes AscI and HpaI and inserted into the AscI and HpaI sites on theCMV driven mammalian expression vector carrying a neomycin selectionmarker. Similarly the FSH alpha DNA was digested with BamHI and NheI andinserted into the sites BamHI and NheI on the expression vector alreadycontaining the FSH beta polypeptide DNA.

The vector DNA was used to transform the DH5α strain of E. coli.Colonies were picked for amplification. Colonies containing the vectorcontaining both FSH alpha and beta were selected for sequencing and allcontained the correct sequences according to SEQ ID NO: 1 and SEQ ID NO:2. Plasmid pFSH A+B#17 was selected for transfection (FIG. 1).

Example 2 Construction of the ST3 Expression Vector

The coding sequence of beta-galactoside alpha-2,3-sialyltransferase 4(ST3, L23767, SEQ ID NO: 3) was amplified by PCR using the primercombination 2,3STfw and 2,3STrev.

2,3STfw (SEQ ID NO: 13) 5′-CCAGGATCCGCCACCATGTGTCCTGCAGGCTGGAAGC-3′2,3STrev (SEQ ID NO: 14) 5′-TTTTTTTCTTAAGTCAGAAGGACGTGAGGTTCTTG-3′

The resulting amplified ST3 DNA was digested with the restrictionenzymes BamHI and AflII and inserted into the BamHI and AflII sites onthe CMV driven mammalian expression vector carrying a hygromycinresistance marker. The vector was amplified as previously described andsequenced. Clone pST3#1 (FIG. 2) contained the correct sequenceaccording SEQ ID NO: 3 and was selected for transfection.

Example 3 Construction of the ST6 Expression Vector

The coding sequence of beta-galactosamide alpha-2,6-sialyltransferase 1(ST6, NM_003032, SEQ ID NO: 4) was amplified by PCR using the primercombination 2,6STfw and 2,6STrev.

2,6STfw (SEQ ID NO: 15) 5′-CCAGGATCCGCCACCATGATTCACACCAACCTGAAG-3′2,6STrev (SEQ ID NO: 16) 5′-TTTTTTTCTTAAGTTAGCAGTGAATGGTCCGG-3′

The resulting amplified ST6 DNA was digested with the restrictionenzymes BamHI and AflII and inserted into the BamHI and AflII sites onthe CMV driven mammalian expression vector carrying a hygromycinresistance marker. The vector was amplified as previously described andsequenced. Clone pST6#11 (FIG. 3) contained the correct sequenceaccording SEQ ID NO: 4 and was selected for transfection.

Example 4 Stable Expression of pFSH α+β in PER.C6® cells. TransfectionIsolation and Screening of Clones.

PER.C6®clones producing FSH were generated by expressing bothpolypeptide chains of FSH from a single plasmid (see Example 1).

To obtain stable clones a liposome based transfection agent with thepFSH α+β construct. Stable clones were selected in VPRO supplementedwith 10% FCS and containing G418. Three weeks after transfection G418resistant clones grew out. Clones were selected for isolation. Theisolated clones were cultured in selection medium until 70-80%confluent. Supernatants were assayed for FSH protein content using anFSH selective ELISA and pharmacological activity at the FSH receptor incloned cell line, using a cAMP accumulation assay. Clones expressingfunctional protein were progressed for culture expansion to 24 well, 6well and T80 flasks.

Studies to determine productivity and quality of the material from sevenclones were initiated in T80 flasks to generate sufficient material.Cells were cultured in supplemented media as previously described for 7days and the supernatant harvested. Productivity was determined usingthe FSH selective ELISA. The isoelectric profile of the material wasdetermined by Isoelectric focusing (IEF), by methods known in the art.Clones with sufficient productivity and quality were selected forsialyltransferase engineering.

Example 5 Level of sialylation is increased in cells that over expressα2,3-sialyltransferase. Stable expression of pST3 in FSH expressingPER.C6® cells; Transfection isolation and screening of clones.

PER.C6® clones producing highly sialylated FSH were generated byexpressing α2,3 sialyltransferase from separate plasmids (Example 2) inPER.C6® cells already expressing both polypeptide chains of FSH (fromExample 4). Clones produced from PER.C6® cells as set out in Example 4were selected for their characteristics including productivity, goodgrowth profile, production of functional protein, and produced FSH whichincluded some sialylation. Stable clones were generated as previouslydescribed in Example 4. Clones were isolated, expanded and assayed. Theα2,3-sialyltransferase clones were adapted to serum free media andsuspension conditions.

As before, clones were assayed using a FSH selective ELISA, functionalresponse in an FSH receptor cell line, IEF, metabolic clearance rate andSteelman Pohley analysis. Results were compared to a commerciallyavailable recombinant FSH (Gonal-f, Serono) and the parental FSHPER.C6®cell lines. FSH produced by most of the clones has significantlyimproved sialylation (i.e. on average more FSH isoforms with highnumbers of sialic acids) compared to FSH expressed without α2,3-sialyltransferase. In conclusion expression of FSH together withsialyltransferase in PER.C6®cells resulted in increased levels ofsialylated FSH compared to cells expressing FSH only.

Example 6 Production and Purification Overview

A procedure was developed to produce FSH in PER.C6®cells that werecultured in suspension in serum free medium. The procedure is describedbelow and was applied to several FSH-producing PER.C6®cell lines.

FSH from α2,3-clone (Example 5) was prepared using a using amodification of the method described by Lowry et al. (1976).

For the production of PER.C6®-FSH, the cell lines were adapted to aserum-free medium, i.e., Excell 525 (JRH Biosciences). The cells werefirst cultured to form a 70%-90% confluent monolayer in a T80 cultureflask. On passage the cells were re-suspended in the serum free medium,Excell 525+4 mM L-Glutamine, to a cell density of 0.3×10⁶ cells/ml. A 25ml cell suspension was put in a 250 ml shaker flask and shaken at 100rpm at 37° C. at 5% CO₂. After reaching a cell density of >1×10⁶cells/ml, the cells were sub-cultured to a cell density of 0.2 or0.3×10⁶ cells/ml and further cultured in shaker flasks at 37° C., 5% CO₂and 100 rpm.

For the production of FSH, the cells were transferred to a serum-freeproduction medium, i.e., VPRO (JRH Biosciences), which supports thegrowth of PER.C6®cells to very high cell densities (usually >10⁷cells/ml in a batch culture). The cells were first cultured to >1×10⁶cells/ml in Excell 525, then spun down for 5 min at 1000 rpm andsubsequently suspended in VPRO medium+6 mM L-glutamine to a density of1×10⁶ cells/ml. The cells were then cultured in a shaker flask for 7-10days at 37° C., 5% CO₂ and 100 rpm. During this period, the cells grewto a density of >10⁷ cells/ml. The culture medium was harvested afterthe cell viability started to decline. The cells were spun down for 5min at 1000 rpm and the supernatant was used for the quantification andpurification of FSH. The concentration of FSH was determined using ELISA(DRG EIA 1288).

Thereafter, purification of FSH was carried out using a modification ofthe method described by Lowry et al. (1976). Purification using chargeselective chromatography was carried out to enrich the highly sialylatedforms by methods well known in the art.

During all chromatographic procedures, enrichment of the sialylatedforms of FSH as claimed herein was confirmed by RIA (DRG EIA 1288)and/or IEF.

Example 7 Quantification of Relative Amounts of α2,3 and α2,6 SialicAcid

The relative percentage amounts of α2,3 and α2,6 sialic acid on purifiedrFSH (Example 6) were measured using known techniques.

N-Glycans were released from the samples using PNGase F underdenaturative conditions and then labelled with 2-aminobenzamide.Released glycan forms were then separated and analysed by Weak AnionExchange (WAX) column for determination of charge distribution. Labelledglycans treated with 2,3,6,8 sialidase for determination of total sialicacid and 2,3 sialidase for determination of 2,3 sialic acid, werefurther analyzed by wax column.

The relative percentages of the charged glycans were calculated fromstructures present in the undigested and digested glycan pools and areshown in FIG. 4 (for 8 samples). These were found to be in the ranges50%-95% (e.g. about 80% to 90%) for α2,3 sialylation and 5% to 50%,generally about 10 to 20% (or about 31% or 35%), for α2,6 sialylation.

Example 8—A multiple dose study investigating FE 999049 in comparison toGONAL-F.

The following describes a randomised, controlled, assessor-blind,parallel groups, multinational, multicentre trial assessing thedose-response relationship of FE 999049 in patients undergoingcontrolled ovarian stimulation for in vitro fertilisation(IVF)/intracytoplasmic sperm injection (ICSI). The patient populationwas 265 IVF patients aged between 18 to 37 years, with BMI 18.5 to 32.0kg/m².

The trial was designed as a dose-response trial with number of oocytesretrieved as the primary endpoint. Secondary endpoints will explore thequalitative and quantitative impact of different doses of FE 999049 withregard to endocrine profile, follicular development, oocytefertilisation, embryo quality and treatment efficiency (i.e. totalgonadotropin consumption and duration of stimulation). The trial isdesigned to evaluate the efficacy of FE 999049 to establish pregnancywhen used in controlled ovarian stimulation for IVF/ICSI cycles.

Subjects were assessed within 3 months prior to randomisation forcompliance with the inclusion and exclusion criteria, including ananti-Müllerian hormone (AMH) assessment to increase homogeneity of thetrial population in relation to ovarian response and minimise the numberof potential poor and hyper-responders to the FE 999049 doses andGONAL-F dose used in the trial. The AMH assessment was measured usingthe AMH Gen-II enzyme linked immunosorbent assay kit (Beckman Coulter,Inc., Webster, Tex.). This assay can detect AMH concentrations greaterthan 0.57 pmol/L with a minimum limit of quantitation of 1.1 pmol/L.

On day 2-3 of their menstrual cycle, subjects were randomised in a1:1:1:1:1:1 fashion to treatment with either 90 IU, 120 IU, 150 IU, 180IU or 210 IU FE 999049 or 150 IU GONAL-F, and ovarian stimulationinitiated. Randomisation was stratified according to AMH level atscreening [5.0-14.9 pmol/L (low AMH) and 15.0 to 44.9 pmol/L (highAMH)).

Gonal-F is filled by mass (FbM) at FDA request; referring to μg dose istherefore appropriate. The Gonal-F label indicates 600 IU/44 μg, whichindicates that 150 IU is 11 μg. However, there is some variation and thebatch certificate for this trial indicated that 11.3 μg Gonal-F wasequivalent to 150 IU. The FE999049 doses are presented by proteincontent (μg) rather than biological activity. Thus the doses of FE999049were 5.2 μg (90 IU), 6.9 μg (120 IU), 8.6 μg (150 IU), 10.3 μg (180 IU)or 12.1 μg (210 IU).

The subject and dose distribution is set out as follows (data are numberof subjects):

TABLE 1 GONAL-F FE 999049 11.3 (11) 5.2 μg 6.9 μg 8.6 μg 10.3 μg 12.1 μgμg Total Screened 334 Randomised 42 45 44 45 46 43 265 and exposed HighAMH 23 26 24 24 26 25 148 strata (56%) (15.0-44.9 pmol/L) Low AMH 19 1920 20 21 18 117 strata (44%) (5.0-14.9 pmol/L) Per-protocol 40 42 42 4444 43 255

The daily dose level of FE 999049 or GONAL-F is fixed throughout theentire stimulation period. During stimulation, subjects are monitored onstimulation day 1, 4 and 6 and hereafter at least every second day. When3 follicles of ≥15 mm are observed, visits are performed daily. Subjectsare treated with FE 999049 or GONAL-F for a maximum of 16 days.

To prevent a premature LH surge, a GnRH antagonist (ganirelix acetate,ORGALUTRAN, MSD/Schering-Plough) may be initiated on stimulation day 6at a daily dose of 0.25 mg and continued throughout the stimulationperiod. Triggering of final follicular maturation is done on the daywhen ≥3 follicles with a diameter ≥17 mm are observed. If there are <25follicles with a diameter ≥12 mm, 250 μg recombinant hCG(choriogonadotropin alfa, OVITRELLE, Merck Serono/EMD Serono) isadministered. If there are 25-35 follicles with a diameter ≥12 mm, 0.2mg GnRH agonist (triptorelin acetate, DECAPEPTYL/GONAPEPTYL, FerringPharmaceuticals) is administered. In case of excessive ovarian response,defined as >35 follicles with a diameter ≥12 mm, the treatment iscancelled. In case of poor ovarian response, defined as <3 follicleswith a diameter ≥10 mm observed on stimulation day 10, the cycle couldbe cancelled.

Oocyte retrieval takes place 36 h (±2 h) after triggering of finalfollicular maturation and the oocytes inseminated by IVF and/or ICSI.Fertilisation and embryo development are assessed from oocyte retrievalto the day of transfer. For subjects who underwent triggering of finalfollicular maturation with hCG, one blastocyst of the best qualityavailable is transferred on day 5 after oocyte retrieval while remainingblastocysts are frozen. For subjects who undergo triggering of finalfollicular maturation with GnRH agonist, no embryo transfer takes placein the fresh cycle and blastocysts are instead frozen on day 5. Vaginalprogesterone tablets (LUTINUS, Ferring Pharmaceuticals) 100 mg 3 timesdaily are provided for luteal phase support from the day after oocyteretrieval until the day of the clinical pregnancy visit. A βhCG test isperformed 13-15 days after embryo transfer and clinical pregnancy willbe confirmed by transvaginal ultrasound (TVU) 5-6 weeks after embryotransfer.

Results

The number of oocytes retrieved (primary endpoint) is shown in thefollowing Table.

TABLE 2 GONAL- FE 999049 F 11.3 5.2 μg 6.9 μg 8.6 μg 10.3 μg 12.1 μg(11)μg Oocytes retrieved All 5.2 (3.3) 7.9 (5.9)  9.2 (4.6) 10.6 (7.0)12.2 (5.9) 10.4 (5.2) High 5.9 (3.9) 9.1 (6.4) 10.6 (4.8) 13.6 (7.8)14.4 (5.8) 12.4 (5.4) AMH Low 4.5 (2.2) 6.3 (4.9)  7.4 (3.8)  6.9 (3.6) 9.4 (4.9)  7.8 (3.4) AMH Data are mean (SD)

The primary objective was met: a significant dose-response relationshipwas established for FE 999049 with respect to number of oocytesretrieved. This finding was observed not only for the overall trialpopulation, but also for each of the two AMH strata used atrandomisation. A significant dose-response for FE 999049 wasdemonstrated for all key objective pharmacodynamic parameters, e.g.estradiol, inhibin B and inhibin A. At a similar microgram dose level,the pharmacodynamic responses with FE 999049 were larger than withGONAL-F (these results not shown).

The serum FSH concentrations after exposure to FE 999049 weresignificantly higher than for GONAL-F. The results confirm that the PKprofile of FE 999049 differs from that of GONAL-F. Fertilisation rates,blastocyst development and pregnancy rates in IVF/ICSI patients treatedwith FE 999049 were within expectations.

There were no safety concerns with the use of FE 999049. A good localtolerability was documented.

Further Analysis

The applicants have further analysed the data to identify the FE 999049dose(s) that fulfil the following criteria with respect to number ofoocytes retrieved:

-   -   Oocytes retrieved in the range 8-14    -   Minimise proportion of patients with <8 oocytes    -   Minimise proportion of patients with <4 or ≥20 oocytes

Low AMH Strata

As seen in Table 2, the dose of FE999049 which fulfilled the firstcriterion (Oocytes retrieved in the range 8-14) was 12.1 μg (mean 9.4oocytes retrieved). The distribution of oocytes is shown in Table 3below.

As shown by the box and arrow, a dose of 12.1 μg FE999049 providesretrieval of the most desirable number of oocytes in 60% of subjects inthe low AMH group. This is a marked improvement on Gonal-F (mostdesirable number of oocytes in only 33% of subjects). There were noindications of early OHSS of a moderate or severe nature and there wereno incidences of preventative action being required; there are noconcerns associated with the dose of 12.1 μg FE999049 in a patienthaving low AMH.

Thus the applicants have found that a dose of, or dose equivalent to, 6to 24 μg, for example 9 to 14 μg, for example 12 μg, human derivedrecombinant FSH is suitable for use in the treatment of infertility in apatient having serum AMH <15 pmol/L, for example 0.05-14.9 pmol/L forexample 5.0-14.9 pmol/L. The dose provides an effective response whileminimising risk of OHSS.

Exploratory Evaluation

As an exploratory evaluation, the present inventors investigated thecontribution of FSH receptor polymorphism on ovarian response andtreatment efficiency following stimulation with FE999049.

Genomic DNA from all patients in the trial was analysed for singlenucleotide polymorphism (SNP) at positions 29, 307 and 680 of the FSH-Rat the University of Modena and Reggio Emilia, Italy. FIG. 4 is aschematic diagram of the FSH Receptor, indicating the position ofpolymorphisms at amino acid positions 307 and 680 of exon 10, andposition 29 in the promoter. This distribution of SNP FSH-R combinationsis as follows: AA 7%, AG 35% and GG 58% for position 29; Thr/Thr 29%,Ala/Thr 54% and Ala/Ala 17% for position 307; and Asn/Asn 30%, Asn/Ser53% and Ser/Ser 17% for position 680. FIG. 5 shows the distribution ofSNP Haplotypes at the FSH receptor gene for the 222 patients treatedwith FSH in the study of Example 8. The distribution for each positionand the overall combinations was not significantly different between thelow AMH and high AMH strata.

The results of the clinical trial were further analysed to assesswhether SNP had any effect on the duration of treatment and total doserequired. This was done for the low AMH group and the high AMH group.

FSHR polymorphism was examined by PCR (Polymerase Chain Reaction) andRFLP (Restriction fragment length polymorphism) by methods known in theart. Women were classified as Asn/Asn, Asn/Ser, and Ser/Ser genotypes.The genetic analysis was described in the following overview protocoland the patients signed a special informed consent. The samples weretaken as part of the other blood samples on stimulation day 1. They weremeasured at the University of Modena and Reggio Emilia.

Overview of Procedures used for SNP-analysis at the FSH Receptor Gene

General Procedures:

-   -   1. Genomic DNA extraction (from blood using Nucleon Genomic DNA        extraction kit, GE HEALTHCARE)    -   2. Operating procedures using nanodrop.

HRM Procedures:

SNPs genotyping by high resolution melting (HRM) methodology (usingSsoFast EvaGreen Supermix cod enzyme. 172-5201, Bio-Rad; HSP-96 plates,cat. HSP9645, Bio-Rad; and CFX96 real-time thermal cycler Bio-Rad.)

Sequencing Procedures:

In case of doubt about HRM results, (after two independent HRM on thesame samples), the following sequencing procedures are utilised:

-   -   1. PCR reaction and amplification.    -   2. PCR product purification.    -   3. Quantification of purified PCR.    -   4. Sequence reaction protocol.    -   5. Sequence product purification.    -   6. Capillary electrophoresis run by ABI PRISM 3130 instrument.    -   7. Assessment and validation of the results obtained by        capillary electrophoresis sequencing with ABI PRISM 3100.

Results

FIG. 7 is a table of results showing, for the full analysis set, theexpected duration of gonadotropin (FSH) treatment (days) delivered topatients/subjects having AMH <15 pmol/L in each of the three distinctFSH receptor genotypes with regard to position 680: Asn/Asn, Asn/Ser andSer/Ser, adjusted for dose.

FIG. 7 shows that the expected mean duration of treatment required forstimulation of a patient having AMH level <15 pmol/L and variant Ser/seris 9.59 days, which is about 1.5 days longer than that required forstimulation of a patient having AMH level <15 pmol/L and variant Asn/Asn(8.13 days), and about 1.3 days longer than that required forstimulation of a patient having AMH level <15 pmol/L and variant Ser/Asn(8.26 days).

As indicated above, success (in terms of pregnancy and/or live birth) ismore likely if the patient has an adequate response (expected ovarianmultifollicular development, rise in circulating 17-β-estradiol)occurring within an ideal treatment window. Success is further enhancedif the response is within the centre of this treatment window; that is,not too early in the window and not too late. Reduction of the durationof treatment in patients having low AMH and variant Ser/Ser at position680 of the FSH receptor (by increasing the dose above 12 μg) may bringthe response towards the centre of the treatment window, with enhancedlikelihood of success.

Accordingly, tailoring the dose to patients identified as having AMHlevel <15 pmol/L and variant Ser/Ser prior to treatment may be possible.Identification of patients having Ser/Ser prior to treatment may allowthe starting dose to be increased in these patients, compared with thosehaving Ser/Asn and Asn/Asn.

As set out above, a dose of 12.1 μg FE999049 provides retrieval of themost desirable number of oocytes in 60% of subjects in the low AMH group(Table 3). The low AMH group shown in Table 3 included patients havingvariant Ser/Ser, as well as those having Ser/Asn and Asn/Asn.Administration of a higher starting dose (for example 9 to 24 pg, forexample >12 to 24 μg, e.g. 12.33 μg or 13 μg human derived recombinant)of FSH to patients having low AMH [AMH level <15 pmol/L, (e.g. 0.05pmol/L to 14.9 pmol/L, e.g. 5.0 pmol/L to 14.9 pmol/L)], as well ashaving variant Ser/Ser at position 680 of the FSH receptor, may beadvantageous because it may provide increased probability of success (interms of pregnancy and/or live birth) and better predictability ofsuccess.

FIG. 7 shows that the mean duration of treatment required forstimulation of a patient having AMH level <15 pmol/L and variant Asn/Asnis 8.13 days, and that required for stimulation of a patient having AMHlevel <15 pmol/L and variant Ser/Asn is 8.26 days, about 1.5 to 1.3 daysshorter than the duration for equivalent treatment of a patient havingAMH level <15 pmol/L and variant Ser/Ser (9.59 days). Thus,identification of patients having AMH level <15 pmol/L and variantSer/Asn and Asn/Asn prior to treatment may allow the starting dose forthese patients to be reduced to less than 12 μg FE 999049 , e.g. 10 to12 μg, or the duration of treatment to be reduced, while still providinga good response in terms of follicular development. This may provide abenefit in terms of cost of the pharmaceutical, and also in terms ofreduction in risk associated with administration of a higher dose thanis required for effect in these patients.

FIG. 6 is a table of results showing, for the full analysis set, theobserved duration of gonadotropin (FSH) treatment (days) and totalgonadotropin (FSH) dose delivered (μg) to patients/subjects having AMH<15 pmol/L in each of the three distinct FSH receptor genotypes withregard to position 680: Asn/Asn, Asn/Ser and Ser/Ser. This confirms theeffects shown in FIG. 7.

The results did not show this effect related to SNP in the high AMHpopulation.

This allows tailoring of the dose of FSH in specific patients identifiedas having specific AMH level, and specific polymorphism at the FSHR.

Example 9—Individualised COS Protocol (Low AMH)

The selected patients are about to undergo COS for in vitrofertilisation (IVF)/intracytoplasmic sperm injection (ICSI) by methodsknown in the art. The pre-treatment protocol includesassessment/screening of the patient's serum AMH using the AMH Gen-IIenzyme linked immunosorbent assay kit (Beckman Coulter, Inc., Webster,Tex.). This assay can detect AMH concentrations greater than 0.57 pmol/Lwith a minimum limit of quantitation of 1.1 pmol/L. AMH may be measuredusing other Assay kits (e.g. available from Roche). The pre-treatmentprotocol includes identification of the allelic variant at position 680of the FSH receptor following extraction of genomic DNA by methods wellknown in the art (e.g. by means of a kit for extraction of genomic DNAfrom blood, and subsequent DNA sequencing, as described in e.g. Gromollet al, Methods, 21, 83-97 (2000), Simoni et al, Journal of ClinicalEndocrinology and Metabolism, Vol 84, No. 2, 751-755 (1999), Falconer etal, Acta Obstet Gynecol Scand 2005: 84: 806-811 (2005), and referencestherein, or by a PCR and RFLP method such as that set out in Loutradiset al, Journal of Assisted Reproduction and Genetics, Vol. 23, No. 4,April 2006).

The COS protocol proceeds in the usual manner apart from administrationof the initial dose of FE 999049 according to AMH level at screening. Apatient with an AMH level of <15 pmol/L and variant Ser/Asn or Asn/Asnwould be administered an initial daily dose of approximately 12 μg FE999049, a human derived recombinant FSH product manufactured accordingto the method of Example 6, or <12 μg FE 999049 , e.g. 10 to 12 μg, e.g.11.33 μg or 11.67 μg, of the human derived recombinant FSH . A patientwith an AMH level of <15 pmol/L and variant Ser/Ser would receive ahigher initial daily dose greater than 12 μg (e.g. 12.33 to 24 μg, or13-24 μg of the human derived recombinant FSH.

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FIGS. 1, 2 and 3: Plasmid maps of the pFSHalpha/beta, pST3 and pST6expression vectors. CMV=Cytomegalovirus promoter, BGHp(A)=Bovine GrowthHormone poly-adenylation sequence, fl ori=fl origin of replication,SV40=Simian Virus 40 promoter, Neo=Neomycin resistance marker,Hyg=Hygromycin resistance marker, SV40 p(A)=Simian Virus 40poly-adenylation sequence, FSH A=Follicle stimulating hormone alphapolypeptide, FSH B=Follicle stimulating hormone beta polypeptide,ST3GAL4=α2,3-sialyltransferase, ST6GAL1=α2,6-sialyltransferase,ColEI=ColEI origin of replication, Amp=ampicillin resistance marker.

Follicle stimulating hormone alpha polypeptide Accession number AH007338Nucleotide sequence of FSH alpha SEQ ID NO: 1 1ATGGATTACT ACAGAAAATA TGCAGCTATC TTTCTGGTCA CATTGTCGGT GTTTCTGCAT 61GTTCTCCATT CCGCTCCTGA TGTGCAGGAT TGCCCAGAAT GCACGCTACA GGAAAACCCA 121TTCTTCTCCC AGCCGGGTGC CCCAATACTT CAGTGCATGG GCTGCTGCTT CTCTAGAGCA 181TATCCCACTC CACTAAGGTC CAAGAAGACG ATGTTGGTCC AAAAGAACGT CACCTCAGAG 241TCCACTTGCT GTGTAGCTAA ATCATATAAC AGGGTCACAG TAATGGGGGG TTTCAAAGTG 301GAGAACCACA CGGCGTGCCA CTGCAGTACT TGTTATTATC ACAAATCTTA AProtein sequence of FSH alpha  (SEQ ID NO: 5) 1MDYYRKYAAI FLVTLSVFLH VLHSAPDVQD CPECTLQENP FFSQPGAPIL QCMGCCFSRA 61YPTPLRSKKT MLVQKNVTSE STCCVAKSYN RVTVMGGFKV ENHTACHCST CYYHKSFollicle stimulating hormone beta polypeptide Accession number NM_000510Nucleotide sequence of FSH beta SEQ ID NO: 2 1ATGAAGACAC TCCAGTTTTT CTTCCTTTTC TGTTGCTGGA AAGCAATCTG CTGCAATAGC 61TGTGAGCTGA CCAACATCAC CATTGCAATA GAGAAAGAAG AATGTCGTTT CTGCATAAGC 121ATCAACACCA CTTGGTGTGC TGGCTACTGC TACACCAGGG ATCTGGTGTA TAAGGACCCA 181GCCAGGCCCA AAATCCAGAA AACATGTACC TTCAAGGAAC TGGTATATGA AACAGTGAGA 241GTGCCCGGCT GTGCTCACCA TGCAGATTCC TTGTATACAT ACCCAGTGGC CACCCAGTGT 301CACTGTGGCA AGTGTGACAG CGACAGCACT GATTGTACTG TGCGAGGCCT GGGGCCCAGC 361TACTGCTCCT TTGGTGAAAT GAAAGAATAA Protein sequence of FSH beta (SEQ ID NO: 6) 1MKTLQFFFLF CCWKAICCNS CELTNITIAI EKEECRFCIS INTTWCAGYC YTRDLVYKDP 61ARPKIQKTCT FKELVYETVR VPGCAHHADS LYTYPVATQC HCGKCDSDST DCTVRGLGPS 121YCSFGEMKE Beta-galactoside alpha-2,3-sialyltransferase 4Accession Number L23767 Nucleotide sequence of ST3GAL4 SEQ ID NO: 3 1ATGTGTCCTG CAGGCTGGAA GCTCCTGGCC ATGTTGGCTC TGGTCCTGGT CGTCATGGTG 61TGGTATTCCA TCTCCCGGGA AGACAGGTAC ATCGAGCTTT TTTATTTTCC CATCCCAGAG 121AAGAAGGAGC CGTGCCTCCA GGGTGAGGCA GAGAGCAAGG CCTCTAAGCT CTTTGGCAAC 181TACTCCCGGG ATCAGCCCAT CTTCCTGCGG CTTGAGGATT ATTTCTGGGT CAAGACGCCA 241TCTGCTTACG AGCTGCCCTA TGGGACCAAG GGGAGTGAGG ATCTGCTCCT CCGGGTGCTA 301GCCATCACCA GCTCCTCCAT CCCCAAGAAC ATCCAGAGCC TCAGGTGCCG CCGCTGTGTG 361GTCGTGGGGA ACGGGCACCG GCTGCGGAAC AGCTCACTGG GAGATGCCAT CAACAAGTAC 421GATGTGGTCA TCAGATTGAA CAATGCCCCA GTGGCTGGCT ATGAGGGTGA CGTGGGCTCC 481AAGACCACCA TGCGTCTCTT CTACCCTGAA TCTGCCCACT TCGACCCCAA AGTAGAAAAC 541AACCCAGACA CACTCCTCGT CCTGGTAGCT TTCAAGGCAA TGGACTTCCA CTGGATTGAG 601ACCATCCTGA GTGATAAGAA GCGGGTGCGA AAGGGTTTCT GGAAACAGCC TCCCCTCATC 661TGGGATGTCA ATCCTAAACA GATTCGGATT CTCAACCCCT TCTTCATGGA GATTGCAGCT 721GACAAACTGC TGAGCCTGCC AATGCAACAG CCACGGAAGA TTAAGCAGAA GCCCACCACG 781GGCCTGTTGG CCATCACGCT GGCCCTCCAC CTCTGTGACT TGGTGCACAT TGCCGGCTTT 841GGCTACCCAG ACGCCTACAA CAAGAAGCAG ACCATTCACT ACTATGAGCA GATCACGCTC 901AAGTCCATGG CGGGGTCAGG CCATAATGTC TCCCAAGAGG CCCTGGCCAT TAAGCGGATG 961CTGGAGATGG GAGCTATCAA GAACCTCACG TCCTTCTGA Protein Sequence of ST3GAL4 (SEQ ID NO: 7) 1MCPAGWKLLA MLALVLVVMV WYSISREDRY IELFYFPIPE KKEPCLQGEA ESKASKLFGN 61YSRDQPIFLR LEDYFWVKTP SAYELPYGTK GSEDLLLRVL AITSSSIPKN IQSLRCRRCV 121VVGNGHRLRN SSLGDAINKY DVVIRLNNAP VAGYEGDVGS KTTMRLFYPE SAHFDPKVEN 181NPDTLLVLVA FKAMDFHWIE TILSDKKRVR KGFWKQPPLI WDVNPKQIRI LNPFFMEIAA 241DKLLSLPMQQ PRKIKQKPTT GLLAITLALH LCDLVHIAGF GYPDAYNKKQ TIHYYEQITL 301KSMAGSGHNV SQEALAIKRM LEMGAIKNLT SFBeta-galactosamide alpha-2,6-sialyltransferase 1Accession number NM_003032 Nucleotide sequence of ST6GAL1 SEQ ID NO: 4 1ATGATTCACA CCAACCTGAA GAAAAAGTTC AGCTGCTGCG TCCTGGTCTT TCTTCTGTTT 61GCAGTCATCT GTGTGTGGAA GGAAAAGAAG AAAGGGAGTT ACTATGATTC CTTTAAATTG 121CAAACCAAGG AATTCCAGGT GTTAAAGAGT CTGGGGAAAT TGGCCATGGG GTCTGATTCC 181CAGTCTGTAT CCTCAAGCAG CACCCAGGAC CCCCACAGGG GCCGCCAGAC CCTCGGCAGT 241CTCAGAGGCC TAGCCAAGGC CAAACCAGAG GCCTCCTTCC AGGTGTGGAA CAAGGACAGC 301TCTTCCAAAA ACCTTATCCC TAGGCTGCAA AAGATCTGGA AGAATTACCT AAGCATGAAC 361AAGTACAAAG TGTCCTACAA GGGGCCAGGA CCAGGCATCA AGTTCAGTGC AGAGGCCCTG 421CGCTGCCACC TCCGGGACCA TGTGAATGTA TCCATGGTAG AGGTCACAGA TTTTCCCTTC 481AATACCTCTG AATGGGAGGG TTATCTGCCC AAGGAGAGCA TTAGGACCAA GGCTGGGCCT 541TGGGGCAGGT GTGCTGTTGT GTCGTCAGCG GGATCTCTGA AGTCCTCCCA ACTAGGCAGA 601GAAATCGATG ATCATGACGC AGTCCTGAGG TTTAATGGGG CACCCACAGC CAACTTCCAA 661CAAGATGTGG GCACAAAAAC TACCATTCGC CTGATGAACT CTCAGTTGGT TACCACAGAG 721AAGCGCTTCC TCAAAGACAG TTTGTACAAT GAAGGAATCC TAATTGTATG GGACCCATCT 781GTATACCACT CAGATATCCC AAAGTGGTAC CAGAATCCGG ATTATAATTT CTTTAACAAC 841TACAAGACTT ATCGTAAGCT GCACCCCAAT CAGCCCTTTT ACATCCTCAA GCCCCAGATG 901CCTTGGGAGC TATGGGACAT TCTTCAAGAA ATCTCCCCAG AAGAGATTCA GCCAAACCCC 961CCATCCTCTG GGATGCTTGG TATCATCATC ATGATGACGC TGTGTGACCA GGTGGATATT 1021TATGAGTTCC TCCCATCCAA GCGCAAGACT GACGTGTGCT ACTACTACCA GAAGTTCTTC 1081GATAGTGCCT GCACGATGGG TGCCTACCAC CCGCTGCTCT ATGAGAAGAA TTTGGTGAAG 1141CATCTCAACC AGGGCACAGA TGAGGACATC TACCTGCTTG GAAAAGCCAC ACTGCCTGGC 1201TTCCGGACCA TTCACTGCTA A 0p-Protein Sequence of ST6GAL1  (SEQ ID NO: 8) 1MIHTNLKKKF SCCVLVFLLF AVICVWKEKK KGSYYDSFKL QTKEFQVLKS LGKLAMGSDS 61QSVSSSSTQD PHRGRQTLGS LRGLAKAKPE ASFQVWNKDS SSKNLIPRLQ KIWKNYLSMN 121KYKVSYKGPG PGIKFSAEAL RCHLRDHVNV SMVEVTDFPF NTSEWEGYLP KESIRTKAGP 181WGRCAVVSSA GSLKSSQLGR EIDDHDAVLR FNGAPTANFQ QDVGTKTTIR LMNSQLVTTE 241KRFLKDSLYN EGILIVWDPS VYHSDIPKWY QNPDYNFFNN YKTYRKLHPN QPFYILKPQM 301PWELWDILQE ISPEEIQPNP PSSGMLGIII MMTLCDQVDI YEFLPSKRKT DVCYYYQKFF 361DSACTMGAYH PLLYEKNLVK HLNQGTDEDI YLLGKATLPG FRTIHC

1-19. (canceled)
 20. A method of treating infertility, comprisingadministering a follicle stimulating hormone (FSH) composition at a doseof or equivalent to from 9 to 24 μg FSH to a patient identified prior totreatment as having variant Ser/Ser at position 680 of the FSH receptor21. The method of claim 20, wherein the patient is identified prior totreatment as having a serum AMH level of 15.0 to 44.9 pmol/L.
 22. Themethod of claim 20, wherein the method comprises daily administration ofthe FSH composition.
 23. The method of claim 20, wherein the methodfurther comprises, prior to administering the FSH composition,identifying the patient as having variant Ser/Ser at position 680 of theFSH receptor.
 24. The method of claim 20, wherein the method comprisesadministering the FSH composition at a dose of or equivalent to fromgreater than 12 to 24 μg FSH.
 25. The method of claim 20, wherein theFSH composition is administered starting on day one of treatment andcontinuing for from six to sixteen days.
 25. The method of claim 20,wherein the FSH is recombinant FSH.
 26. The method of claim 20, whereinthe FSH is recombinant FSH that includes α2,3-sialylation andα2,6-sialylation.
 27. The method of claim 20, wherein the FSH isrecombinant FSH that includes α2,3-sialylation and α2,6-sialylation,wherein from 1 to 99% of the total sialylation is α2,6-sialylation andfrom 99% to 1% of the total sialylation is α2,3-sialylation.
 28. Themethod of claim 20, wherein the FSH is recombinant FSH that includesα2,3-sialylation and α2,6-sialylation, wherein from 1 to 50% of thetotal sialylation is α2,6-sialylation and from 50% to 99% of the totalsialylation is α2,3-sialylation.
 29. A method of treating infertility,comprising administering a follicle stimulating hormone (FSH)composition at a dose of or equivalent to from 10 to 12 μg FSH to apatient identified prior to treatment as having variant Asn/Asn orvariant Asn/Ser at position 680 of the FSH receptor.
 30. The method ofclaim 29, wherein the patient is identified prior to treatment as havinga serum AMH level of 15.0 to 44.9 pmol/L.